Raf-1 is a serine/threonine protein kinase that plays a central role in signal transduction from the membrane to the nucleus. Activated Raf-1 activates transcription from AP-1, Ets, and NF-kappaB binding sites. Raf- induced transactivation from the NF-kappaB binding sites in the HIV long terminal repeat (LTR) appears to result from Raf-activating GABP, an ets family transcription factor that is composed of two subunits (alpha and beta). Overexpression of GABP induced expression from the HIV long terminal repeat (LTR) and activated Raf synergized with GABP for HIV-LTR driven expression. Using a gel shift assay we have determined that purified GABP binds to the NF-kappaB binding sites in the HIV-LTR in vitro. This binding was specific and required both NF-kappaB binding sites since a single site failed to compete for binding. To determine if activated Raf phosphorylates GABP directly, we used activated Raf produced by mixed infections in Sf9 cells with baculoviruses expressing Raf-1, v- Ras and Lck. Although this activated Raf efficiently phosphorylated mitogen-activated protein (Map) kinase kinase initiating a kinase cascade which resulted in Jun phosphorylation by Map kinase, direct GABP phosphor- ylation was not observed. Activated Map kinase, however, did phos- phorylate GABP alpha and beta1 subunits in vitro. Moreover, activated Raf-1 appears to induce the phosphorylation of GABP beta1 in vivo. We are currently determining the physiological consequences of these phosphorylation events.